Project Summary The Centers for Disease Control and Prevention have stated that insufficient sleep is an epidemic that poses significant clinical and economic impacts. Poor sleep can be caused by impaired sleep homeostasis which regulates sleep need as a function of prior wakefulness. Therefore, determining the cellular basis of sleep homeostasis is necessary to understand the underlying causes of abnormal sleep. The biological substrates of sleep homeostasis are incompletely understood, but neuronal-glial feedback mechanisms may play a central role. Because neuronal and astroglial chemical signaling is mediated, in part, by intracellular calcium waves, we hypothesize that neuronal and astroglial intracellular calcium dynamics contribute to the accumulation and discharge of sleep need. This hypothesis will be tested using a multimodal imaging approach to measure in vivo intracellular calcium activity in neuronal and astroglial somata and processes. Genetically encoded calcium indicators will be selectively expressed in neurons or in astrocytes to assess intracellular calcium dynamics using two microscopy methodologies in the same, unanesthetized mouse: 1) a lightweight, head-mounted epifluorescent microscope that permits free movement and behavior and 2) two-photon microscopy combined with a platform that allows for head-restrained cage navigation. Cellular calcium dynamics will be simultaneously recorded with sleep-wake behavior, as determined by electroencephalography and electromyography, under physiological conditions and in response to sleep deprivation. Thus, this project will develop a scalable platform methodology for the functional assessment of different brain cell types in freely behaving and unanesthetized animals. Our approach integrates complementary microscopy techniques with standard behavioral analyses for the assessment of neuronal and glial activity during complex processes and behavior. As such, the proposed design can be extended to studies of addiction, aging, learning and memory, and more to further elucidate the underlying neurobiology of centrally mediated processes and behaviors.